Hepatocellular carcinoma, a fatal liver cancer, affects 600 000 people annually and ranks third in cancer-related lethality. In this work we report the synthesis and related biological activity of novel dihydropyrimidones. Among the tested compounds, 5-acetyl-4-(1H-indol- 3-yl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one (4g) was found to be most active towards the HepG2 cell line (IC50 = 17.9 mu M), being at the same time 7.6-fold selective over normal (LO2) liver cells (IC50 = 136.9 mu M). Subsequently, we identified peroxisome proliferator-activated receptor gamma as a target of compound 4g using an in silico approach, and confirmed this mode-of-action experimentally

Alan Prem Kumar, .

Andreas Bender, .

Basappa, .

Dinesh, K.R.

Gautam Sethi, .

Hanumantharayappa Bharathkumar, .

Julian, E.Fuchs.

Kodappully Sivaraman Siveen, .

Mohan, C.D.

Nima Mohandas, .

Rangappa, K.S.

Shardul Paricharak, .

Shobith Rangappa, .

ePrints@Bangalore University

RSC AdvancesCOMMUNICATIONPublished on 02 September 2014. Downloaded by York University on 23/10/2014 14:03:27. View Article OnlineView Journal  | View IssueSynthesis, biologaLaboratory of Chemical Biology, DepartmPalace Road, Bangalore 560001, India. E-mbUnilever Centre for Molecular ScienceUniversity of Cambridge, Lenseld Road, CBcDivision of Medicinal Chemistry, Leiden AcUniversity, P.O. Box 9502, 2300 RA Leiden,dDepartment of Pharmacology, Yong Loo LinSingapore, Singapore 117597, SingaporeeInterdisciplinary Research Group of InfectiResearch & Technology Centre (SMART), 13fCancer Science Institute of Singapore, Nat117599, SingaporegDepartment of Chemistry, University of MIndiahSchool of Biomedical Sciences, Faculty of HAustralia, AustraliaiDepartment of Biological Sciences, Universi† Electronic supplementary information (Eeducts and products as well as syncharacterization of the compounds10.1039/c4ra08713eCite this: RSC Adv., 2014, 4, 45143Received 15th August 2014Accepted 2nd September 2014DOI: 10.1039/c4ra08713ewww.rsc.org/advancesThis journal is © The Royal Society of Cical evaluation and in silico and invitro mode-of-action analysis of noveldihydropyrimidones targeting PPAR-g†Hanumantharayappa Bharathkumar,a Shardul Paricharak,bc K. R. Dinesh,aKodappully Sivaraman Siveen,d Julian E. Fuchs,b Shobith Rangappa,ef C. D. Mohan,gNima Mohandas,d Alan Prem Kumar,dfhi Gautam Sethi,d Andreas Bender,*b Basappa*aand K. S. Rangappa*gHepatocellular carcinoma, a fatal liver cancer, aﬀects 600 000 peopleannually and ranks third in cancer-related lethality. In this work wereport the synthesis and related biological activity of novel dihy-dropyrimidones. Among the tested compounds, 5-acetyl-4-(1H-indol-3-yl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one (4g) was foundto bemost active towards the HepG2 cell line (IC50¼ 17.9 mM), being atthe same time 7.6-fold selective over normal (LO2) liver cells (IC50 ¼136.9 mM). Subsequently, we identiﬁed peroxisome proliferator-acti-vated receptor g as a target of compound 4g using an in silicoapproach, and conﬁrmed this mode-of-action experimentally.The peroxisome proliferator-activated receptor (PPAR) belongsto the nuclear hormone receptor superfamily of transcriptionfactors that includes 48 human transcription factors. Its activityis regulated by direct binding of steroid and thyroid hormones,vitamins, lipid metabolites, and xenobiotics.1 PPARs hetero-dimerize with retinoid X receptors, which get activated and bindto specic response elements in the target DNA of various targetent of Chemistry, Bangalore University,ail: salundibasappa@gmail.comInformatics, Department of Chemistry,2 1EW, Cambridge, UKademic Centre for Drug Research, LeidenThe NetherlandsSchool of Medicine, National University ofous Diseases, Singapore-MIT Alliance for8602, Singaporeional University of Singapore, Singaporeysore, Manasagangotri, Mysore 560001,ealth Sciences, Curtin University, Westernty of North Texas, Denton, Texas, USASI) available: Chemical structures of allthesis protocols and spectroscopicare available as ESI. See DOI:hemistry 2014genes,2,3 PPARs consist of three diﬀerent isoforms, PPAR-a,PPAR-b/d, and PPAR-g. Due to their central function in a varietyof physiological processes, PPARs are important targets in drugdiscovery.4 Small molecules frequently interact with more thana single PPAR isoform leading to unique proles of their bio-logical eﬀects.5In particular, PPAR-g has been established as a key regulatorfor inammation,6 proliferation,7 metabolism,8 and diﬀerenti-ation,9 and it is also upregulated by many tumor suppressorgenes.10 PPAR-g is overexpressed in several types of humancancers, including breast, colon, bladder, and prostate cancer,and it also induces apoptosis in several malignant cell line-ages.11 Furthermore, PPAR-g ligands such as 15d-PGJ2 as well asantidiabetic thiazolidinediones (TZDs) function as anti-prolif-erative and proapoptotic agents, suggesting that PPAR-g couldbe a promising therapeutic target for the treatment ofcancer.12–14 Non-thiazolidinedione derivatives, such as 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, GW-7845, JTT-501, KRP-297, L-764406, MCC-555 and GW-0207 are further knownsynthetic ligands of PPAR-g.15On the other hand, the natural product ectoine, a tetrahy-dropyrimidine derivative, oﬀers protection against the eﬀects ofischemia-reperfusion injury in intestinal transplantation in vivo.16()-Aplicyanin analogs bearing a pyrimidone moiety showedsignicant anti-tumor activity via targeting p38 MAP kinase.17–19Similar small molecules, dihydropyrimidines (DHPs), havealso been reported as anti-cancer agents.20 Recently, pyrimi-dine-tethered curcumins showed anticancer activity by target-ing EGFR tyrosine kinase.21In continuation of our interest to synthesize novel bioactiveheteocycles,22–24 we herein report the synthesis of DHP tetheredto various functional heterocycles like indole, avone andbenzofuran. Furthermore, and conceptually rather novel, werationalized the mode-of-action for the lead DHP as PPAR-g viaan in silico cheminformatics approach, followed by experi-mental validation. Details on synthesis, compound chararacte-rization, biological assays, and in silico methods are describedin more detail in the ESI.†RSC Adv., 2014, 4, 45143–45146 | 45143RSC Advances CommunicationPublished on 02 September 2014. Downloaded by York University on 23/10/2014 14:03:27. View Article OnlineFor synthesis, multi-component reactions (MCRs) have beenemployed which constitute an eﬃcient synthetic strategy forrapid and eﬀective laboratory organic transformations, becauseproducts are prepared in a one-pot and single step approach andthe diversity can be obtained directly by changing the reactingcomponents.25 Here, we synthesized the dihydropyrimidonebearing small molecules via multi-component Biginelli reac-tion.26 The mechanism for the formation of the title compoundsinvolves the condensation between the aldehyde and urea toform an iminium intermediate, which acts as electrophile for thenucleophilic addition of the ketoester enol, and the ketonecarbonyl of the resulting adduct undergoes condensation withthe amine group of urea to give the cyclized product (Fig. 1A).Also further heterocycles such as indole, avone and benzofuranmoieties were successful employed for the preparation of noveldihydropyrimidones (see ESI Table 1†). The protocol was eﬀec-tive with aromatic amines having electron donating groups, andproducts were identied based on IR, LCMS, 1H NMR, and 13CNMR spectra (see ESI†). In the next step, the cytotoxic eﬀects ofdihydropyrimidones was investigated in HepG2 cell lines usingthe MTT assay. The cells were treated with 0, 10, 25, 50, and 100mM concentrations of dihydropyrimidones for 72 h. DHPs werefound to inhibit the viability of HepG2 cells in a dose-and time-dependent manner. Compound 4g, 4k and 4p were found to bemost eﬀective with IC50 values less than 50 mM. In addition, wewere able to identify that all the dihydropyrimidone series ofcompounds inhibited the viability of immortalized normalhuman liver cells, LO2, at higher concentrations. Compound 4gwas found have an IC50 value of 17.9 mM against HepG2 and hasthe highest, 7.6-fold selectivity over the normal LO2 liver cell line(IC50 ¼ 136.93 mM; see ESI Table 2† for more details). Therefore,we considered 4g (Fig. 1B) in more detail and studied its mode-of-action in HepG2 cells.Fig. 1 Synthetic scheme of novel dihydropyrimidones and cytotoxicitystudies for the lead compound 4g against various HCC cells.45144 | RSC Adv., 2014, 4, 45143–45146Human protein targets were predicted for the most bioactivecompound 4g, and of all predicted targets (ESI Table 3†), PPAR-g was the only target with score of 26.02, exceeding the signif-icance cut-oﬀ of 10. Given the involvement of PPAR-g in theinduction of apoptosis and cancer development, as described inprevious studies,4–6 this target was selected for further analysis.PPAR-g has been extensively shown to be associated withanti-cancer eﬀects in a variety of cancer types including HCC.HepG2 cells were treated with diﬀerent concentrations ofcompound 4g for 8 h and then analyzed for the expression ofPPAR-g by western blot analysis. It was found that compound 4gcould substantially increase PPAR-g expression in a dose-dependent manner, with maximum eﬀect at 15 mM concentra-tion (Fig. 2A). Compound 4g also increased PPAR-g expressionin a time-dependent manner, with maximum activity at 8 h(Fig. 2B). Evidently, the DNA-binding assay for PPAR-g innuclear extracts showed that compound 4g signicantlyenhanced PPAR-g DNA binding ability in a time dependentmanner (Fig. 2C).DHPs are well known ligands for PPAR-g. In order to deter-mine the interaction of dihydropyrimidones towards PPAR-g,the co-crystal structure of human PPAR-g in complex withrosiglitazone was considered for our studies.27 Using DiscoveryStudio (DS) default tools and settings, the ligand bindingdomain (LBD) of PPAR-g was identied and ligands wereprepared for docking utilizing the CDOCKER programme of theligand–receptor interaction module of Discovery Studio version2.5, the dihydropyrimidones were docked into the LBD of PPAR.The CDOCKER energies for all docked ligands (ESI Table 4†)indicate that compound 4g bound to the LBD region with thelowest (and thus most favorable) score of 24.0 kcal mol1 (ESITable 3†). Furthermore, the binding pose of compound 4g wasfound to overlap with the hydrophobic tail of TZDs (rosiglita-zone; Fig. 2D). Compound 4g forms mainly hydrophobiccontacts with residues at the LBD region of PPAR-g includingFig. 2 In silico and in vitro rationalization of the mode-of-actionanalysis for the lead compound 4g that targets PPAR-g in HepG2 cells.This journal is © The Royal Society of Chemistry 2014Communication RSC AdvancesPublished on 02 September 2014. Downloaded by York University on 23/10/2014 14:03:27. View Article OnlineIle281, Met348, Ile353, Val339, Cys285, Ile341, Ser342, Gly284,Tyr321, Ile326, Lys367, Phe363, Met364, Leu330, Ser289, andArg288 (Fig. 2E). Thereby, CH–p interactions with Leu-330 andvan der Waals contacts with the alkyl chain of Arg-288 areproposed as main driving forces of the ligand. This moleculardocking study revealed that compound 4g could be a suitablenon-TZD ligand for PPAR-g.We investigated whether activation of PPAR-g in HepG2 cellsby compound 4g leads to apoptosis. In HepG2 cells treated withcompound 4g, there was a time-dependent reduction in thelevels of procaspase-3 (Fig. 3A). This suggests that cleavageevents had occurred, indicating the activation of caspase-3, thelevels of which were shown to be increased aer 48 h. Activationof caspase-3 led to the cleavage of a 118 kDa Poly (ADP-ribose)polymerase (PARP) protein into an 85 kDa fragment (Fig. 3B).This result clearly suggests that compound 4g induces caspase-3-dependent apoptosis in HepG2 cells. In addition, compound4g was found to downregulate the anti-apoptotic and prolifer-ative proteins, Bcl-2 and Cyclin D1 in a time dependent manner(Fig. 3C), with maximum eﬀect observed at 48 h. Pronouncedexpression of PPAR-g was demonstrated in HCC cells treatedwith rosiglitazone and such induction markedly suppressed themigration of HCC cells.28 The eﬀect of compound 4g on themigration of HepG2 cells was investigated using a woundhealing assay. We found that compound 4g treatment signi-cantly suppressed the migration of HepG2 cells, and thepretreatment with GW9662, a pharmacological PPAR-g inhib-itor, reversed the anti-migratory eﬀects of compound 4g asshown in Fig. 3D and E. It is well known that PPAR-g ligandsmay have therapeutic value for the treatment of highly invasivebreast cancer by targeting invasion.29 An in vitro Bio-CoatMatrigel invasion assay was performed (BD Biosciences, SanJose, CA, USA), and it was found that upon treatment withcompound 4g there was a signicant reduction in the numberof cells that could invade the Matrigel coated polycarbonateFig. 3 Apoptotic induction, anti-migration, and anti-invasive activityof the compound 4g in HCC cells.This journal is © The Royal Society of Chemistry 2014membrane, indicating that compound 4g could indeed signi-cantly inhibit the invasive property of HepG2 cells (Fig. 3F andG). Moreover, we found that pretreatment with the PPAR-gantagonist GW9662 reversed the anti-invasive potential ofcompound 4g in HCC cells, conrming that the activity ismediated through inhibition of PPAR-g pathway.In conclusion, we herein report the synthesis of novel dihy-dropyrimidones and found 5-acetyl-4-(1H-indol-3-yl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one (4g) to be most active againstHCC cells. Furthermore, compound 4g down-regulated anti-apoptotic and proliferative proteins such as Bcl-2 and Cyclin D1in a time-dependent manner. In silico studies predicted PPAR-gas mode-of-action of compound 4g, which could be validated byin vitro studies. Molecular docking furthermore suggested thebinding pose of 4g within the ligand binding domain of PPAR-g,which overlaps with the tail of rosiglitazone. Compound 4ginhibited the invasive property of HepG2 cells and thepretreatment with GW9662 reversed the anti-invasive potentialof compound 4g in HCC cells in vitro, thereby conrming thatthe activity is mediated through inhibition of PPAR-g pathway.Hence, both in silico and experimental methods agree on themode-of-action of compound 4g via PPAR-g.AcknowledgementsThis research was supported by the Department of Science andTechnology (no. SR/FT/LS-142/2012), University GrantsCommission (41-257-2012-SR), and Vision Group Science andTechnology to Basappa. Basappa thanks Karnataka University,INDIA for DC PAVATE Fellowship and IISc, Bangalore forproviding access to the NMR Facility. Bharathkumar thanksUGC for a BSR fellowship. SP thanks the Netherlands Organi-sation for Scientic Research (NWO, grant number NWO-017.009-065) and the Prins Bernhard Cultuurfonds for funding.CDM thanks DST for INSPIRE fellowship.Notes and references1 M. Lehrke and M. A. Lazar, Cell, 2005, 123, 993–999.2 F. S. 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Synthesis, biological evaluation and in silico and in vitro mode-of-action analysis of novel dihydropyrimidones targeting PPAR-gamma

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Synthesis, biological evaluation and in silico and in vitro mode-of-action analysis of novel dihydropyrimidones targeting PPAR-gamma

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